Analyte detection and quantification is important in many different technical areas such as biomedical research (both in industry and in academia), clinical diagnostics, agricultural diagnostics and practices, environmental control, forensics, personalized medicine, pharmacogenomics, and others.
While some analytes can be detected and measured directly, very often it is useful to detect analytes using attached labels that can be more easily detected and measured. Multiple labels have been developed over the years for various analytical applications: radioisotopes, fluorescent dyes and quantum dots have all been used in analyte labeling. The signal generated by such labels can be detected with appropriate instrumentation. For example, the light emitted by fluorescent dyes upon suitable excitation can be detected and quantified with light measuring devices such as photomultipliers (PMT) or quantitative charge-coupled devices (CCDs).
When the analyte is contained in a complex mixture it needs to be specifically targeted for labeling. This is often accomplished through analyte-specific probes, such as antibodies. For example, when an analyte-specific antibody which is labeled with a fluorescent dye binds to the analyte of interest, the fluorescence associated with the antibody indirectly quantifies the amount of analyte present. Similarly, when an isotopically-labeled DNA probe that is complimentary to an analyte mRNA binds to its target analyte, the radioactivity level that is detected corresponds to the amount of the specific mRNA that is assayed. In these types of applications, the analyte is often referred to as the “target”.
In most analytical methods using detectable labels, multiple analyte molecules generate an aggregate signal that is detected simultaneously. Such a mode of quantification is referred to as analog, since it measures an analog value, such as light intensity or level of radioactivity. In contrast, digital techniques would detect each individual molecule (Ishijima and Yanagida 2001) of the analyte separately and tally their counts. Digital techniques offer multiple advantages over analog ones: high sensitivity, high signal-to-noise, high accuracy, and low cost. These advantages stem from the fact that the measurement system does not normally require a precisely calibrated linear response and large dynamic range, as its goal is to only detect the presence or absence of a particular molecule, while the quantification is achieved through tallying the number of times the molecule has been detected (Dimitrov 2001).
Multiparameter or multiplexed detection and quantification simultaneously measures multiple and different species of analyte. For example, in expression profiling, mRNA levels for thousands of different genes can be assayed simultaneously (Schena 2003). Distinct labels for such a large diversity of analytes can be generated through combinatorial assembly of a limited number of detectable elements. Such labels can be called barcodes because they exhibit some aspects of barcode labels widely applied in the retail industry and elsewhere, although they are functionally very different from such barcode labels.